Since the immune system is central to the protection of an individual from an external biological threat, diseases of the immune system are therefore a consequence of one or a combination of three problems with the immune system.                Underproduction or suppression of the immune system (e.g. AIDS or SIDS);        Overproduction of cells of the immune system (e.g. Leukemia or Lymphoma);        Overproduction of the effects of the immune system (e.g. Inflammation);        Inappropriate activation of the effects of the immune system (e.g. allergy).        
Treatments of diseases of the immune system are therefore aimed at either the augmentation of immune response or the suppression of inappropriate responses. Since cytokines play a pivotal role in the regulation of the immune system, they are appropriately considered to be key targets for therapeutic intervention in immune pathologies. Similarly, the intracellular signal transduction pathways that are regulated by cytokines are potential points of therapeutic intervention in diseases that involve overproduction of cytokine signaling. The JAK family of protein tyrosine kinases (PTKs) play a central role in the cytokine dependent regulation of the proliferation and end function of several important cell types of the immune system. As such they represent excellent, well-validated targets for the purpose of drug discovery; the notion being that potent and specific inhibitors of each of the four JAK family members will provide a means of inhibiting the action of those cytokines that drive immune pathologies, such as asthma (e.g. IL-13; JAK1, JAK2), and leukemia/lymphoma (e.g. IL-2: JAK1 and JAK3).
Furthermore, certain types of cancer such as prostate cancer develop autocrine production of certain cytokines as a selectable mechanism of developing growth and/or metastatic potential. An example of this is cancer of the prostate, where IL-6 is produced by and stimulates the growth of prostate cancer cell lines such as TSU and TC3 (Spiotto M T, and Chung T D, 2000). Interestingly, levels of IL-6 are elevated in sera of patients with metastatic prostate cancer.
A great deal of literature covers the area of cytokine signaling. The present inventors have focused on the JAK/STAT pathway that is involved in the direct connection of cytokine receptor to target genes (such as cell cycle regulators (e.g. p21) and anti-apoptosis genes (such as Bcl-XL)).
The JAK/STAT Pathway
The delineation of a particularly elegant signal transduction pathway downstream of the non-protein tyrosine kinase cytokine receptors has recently been achieved. In this pathway the key components are: (i) A cytokine receptor chain (or chains) such as the Interleukin-4 receptor or the Interferon γ receptor; (ii) a member (or members) of the JAK family of PTKs; (iii) a member (s) of the STAT family of transcription factors, and (iv) a sequence specific DNA element to which the activated STAT will bind.
The general principles of the JAK/STAT pathway are shown below, for the IFNγ receptor, an example of the class II cytokine receptors. Although the same basic mechanism is initiated by each family of cytokine receptors, there remain discrepancies in detail which are at present unresolved, although they presumably define the specificity of the cellular response to particular cytokines.
A review of the JAK/STAT literature offers strong support to the notion that this pathway is important for the recruitment and marshalling of the host immune response to environmental insults, such as viral and bacterial infection. This is well exemplified in Table 1 and Table 2. Information accumulated from gene knock-out experiments have underlined the importance of members of the JAK family to the intracellular signaling triggered by a number of important immune regulatory cytokines (Table 7).
The therapeutic possibilities stemming from inhibiting (or enhancing) the JAK/STAT pathway are thus largely in the sphere of immune modulation, and as such are likely to be promising drugs for the treatment of a range of pathologies in this area. In addition to the diseases listed in Tables 1 and 2, inhibitors of JAKs could be used as immunosuppressive agents for organ transplants and autoimmune diseases such as lupus, multiple sclerosis, rheumatoid arthritis, Type I diabetes, autoimmune thyroid disorders, Alzheimer's disease and other autoimmune diseases. Additionally, treatment of cancers such as prostate cancer by JAK inhibitors is indicated.
TABLE 1Cell TypesDisease TypeInvolvedCharacteristicsAtopyAllergic Asthma(Mast Cells)T-cell activation ofAtopic Dermatitis(EosinophilsB-cells followed by(Eczema)(T-CellsIgE mediated activationAllergic Rhinitis(B-Cellsof resident Mast cellsand EosinophilsCell MediatedHypersensitivityAllergic Contact(T-cellsT-cellDermatitis(B-cellshypersensitivityHypersensitivityPneumonitisRheumatic DiseasesSystemic LupusErythematosus (SLE)Rheumatoid Arthritis(Monocytes)Cytokine ProductionJuvenile Arthritis(Macrophages(e.g. TNF, IL-1,Sjögren's Syndrome(NeutrophilsCSF-1, GM-CSF)Scleroderma(Mast CellsT-cell ActivationPolymyositis(EosinophilsJAK/STAT activationAnkylosing Spondylitis(T-CellsPsoriatic Arthritis(B-CellsViral DiseasesEpstein Barr VirusLymphocytesJACK/STAT Activation(EBV)Hepatitis BHepatocytesJACK/STAT ActivationHepatitis CHepatocytesJACK/STAT InhibitionHIVLymphocytesJACK/STAT ActivationHTLV 1LymphocytesJACK/STAT ActivationVaricella-ZosterFibroblastsJACK/STAT InhibitionVirus (VZV)Human PapillomaEpithelial cellsJACK/STAT InhibitionVirus (HPV)CancerLeukemiaLeucocytes(Cytokine productionLymphomaLymphocytes(JAK/STAT Activation
There are many different types of protein kinase. Each type has the ability to add a phosphate group to an amino acid in a target protein. The phosphate is provided by hydrolyzing ATP to ADP. Typically, a protein kinase has an ATP-binding site and a catalytic domain that can bind a portion of the substrate protein. The JAK family of protein tyrosine kinases (PTKs) play a central role in the cytokine dependent regulation of the proliferation and end function of several important cell types of the immune system.
The JAK family of Protein Tyrosine Kinases (PTKs) represent excellent drug discovery targets for the following reasons:                They are proven key players in the cellular response to a number of important cytokines (from gene Knock-out and biochemical studies);        Whilst each of the JAK family members are relatively widely expressed, their PTK activity is activated only at sites where cytokine levels are relatively high, i.e. at a local site of inflammation;        They are enzymes permitting effective inhibition of signal amplification and facilitating drug design;        
Therapeutic applications in which inhibitors of particular JAK kinases may be useful are outlined in Table 2 below:
TABLE 2Diseases Potentially Treatable By JAK-Based Drug TherapiesJAK familyStrength ofTarget DiseaseCytokinememberAssociationAsthmaIL-4 &JAK1 & JAK3+ + +IL-9IL-13JAK1 & JAK2+ + +IL-5JAK 2+ + +EczemaIL-4JAK1 & JAK3+ + +IFN-αJAK1 & JAK2+ + +Food AllergyIL-4JAK1 & JAK3+ + +Inflammatory BowelIL-4JAK1 & JAK3+ + +Disease & Crohn'sDiseaseLeukaemia And(IL-2)JAK3, JAK1 &+ + +LymphomaJAK2Cutaneous InflammationGM-CSF &JAK1 & JAK2+ + +IL-6Immune Suppression ByIL-10JAK1 & TYK2+ + +Solid TumourMultiple MyelomaIL-6JAK1, JAK2 &+ + +TYK 2
TABLE 3A list of Cytokines that use theJAK/STAST pathway for SignalingCYTOKINEJAK1JAK2JAK3TYK2IL-2, IL-4, IL-7, IL-9, IL15 (IL-13)+(+)+(+)IL-13++(+)IL-3, IL-5, GM-CSF+IL-6, IL-11, OSM, CNTF, LIF+++IL-12Leptin+GH, PRL, Epo, Tpo+IFNα, IFNβ, IL-10++IFNγ++
A direct comparison of the four mammalian JAK family members revealed the presence of seven highly conserved domains (Harpur et al., 1992). In seeking a nomenclature for the highly conserved domains characteristic of this family of PTKs, the classification used herein was guided by the approach of Pawson and co-workers (Sadovski et al., 1986) in their treatment of the SRC homology (SH) domains. The domains have been enumerated accordingly with most C-terminal homology domain designated JAK Homology domain 1 (JH1). The next domain N-terminal to JH1 is the kinase-related domain, designated here as the JH2 domain. Because of its overall similarity to other kinase domains it is also known as the Kinase-Like Domain or KLD. Each domain is then enumerated up to the JH7 located at the N-terminus (FIG. 1 shows a schematic representation of this nomenclature). The high degree of conservation of these JAK homology (JH) domains suggests that they are each likely to play an important role in the cellular processes in which these proteins operate. However, the boundaries of the JAK homology domains are arbitrary, and may or may not define functional domains. Nonetheless, their delineation is a useful device to aid the consideration of the overall structural similarity of this class of proteins.
The PTK Domain
The feature most characteristic of the JAK family of PTKs is the possession of two kinase-related domains (JH1 and JH2/KLD) (Wilks et al., 1991). The putative PTK domain of JAK1 (JH1) contains highly conserved motifs typical of PTK domains, including the presence of a tyrosine residue at position 1022 located 11 residues C-terminal to sub-domain VII that is considered diagnostic of membership of the tyrosine-specific class of protein kinases. Alignment of the human JAK1 PTK domain (255 amino acids), with other members of the PTK class of proteins revealed homology with other functional PTKs (for example, 28% identity with c-fes (Wilks and Kurban, 1988) and 37% homology to TRK (Kozma et al., 1988). The JH1 domains of each of the JAK family members possess a interesting idiosyncrasy within the highly conserved sub-domain VIII motif (residues 1015 to 1027 in JAK2, SEQ ID NO:1) that is believed to lie close to the active site, and define substrate specificity. The phenylalanine and tyrosine residues flanking the conserved tryptophan in this motif are unique to the JAK family of PTKs (see Table 4). Aside from this element, the JH1 domains of each of the members of the JAK family are typical PTK domains.
TABLE 4Motif VIII of the JAK family of PTKsbears a conserved tyrosineMotif VIIJAK1DSPVFWYAPECLI (SEQ ID NO: 2) JAK2ESPIFWYAPESLT (SEQ ID NO: 3) Tyk2DSPVFWYAPECLK (SEQ ID NO: 4) JAK3QSPIFWYAPESLS (SEQ ID NO: 5) EGF-RKVPIKWMALESIL (SEQ ID NO: 6) c-SRCKFPIKWTAPEAAL (SEQ ID NO: 7)
The Kinase-like Domain (KLD or JH2 Domain)
Based upon cladograms generated using programmes such as Pile Up, the second kinase-like domain (KLD or JH2 Domain) is clearly ancestrally related to the broader family of kinase domains, by virtue of the presence of most of the key kinase motifs defined by Hanks, Quinn and Hunter (Hanks et al., 1988; Hanks & Quinn 1991). However, in order to distinguish the KLD domain motifs from the PTK domain motifs, they have been assigned a subscript a, (e.g Ia, IIa, IIIa etc.) with respect to their similarity to the sub-domains described by Hanks and co-workers (Hanks et al., 1988).
The overall sequence similarity of this domain to the kinase domains of both the PTK and serine/threonine kinase families implies that this region of the protein might also function as a protein kinase. There are, however, significant differences in the sequences of key motifs within this domain which suggest that the catalytic activity of the KLD domain may be something other than serine/threonine or tyrosine phosphorylation or indeed may not be kinase related. For example, comparison of sub-domain VIa of the KLD domain with sub-domain VI of members of the PTK and Serine/Threonine families shows the replacement of a conserved acidic amino acid (aspartic acid) with a neutral amino acid (asparagine). For example,
TABLE 5Motif VIb of the KLD of the JAK family ofPTK1 bears a conserved Asparagine residueMotif VIbJAK1(KLD)VHGNVCTKNLL (SEQ ID NO: 8)  JAK2(KLD)IHGNVCAKNIL (SEQ ID NO: 9)  Tyk2(KLD)VHGNVCGRNIL (SEQ ID NO: 10) JAK3(KLD)PHGNVSARKVL (SEQ ID NO: 11) JAK1(JH1)VHRDLAARNVL (SEQ ID NO: 12) EGF-RVHRDLAARNVL (SEQ ID NO: 13) cAMPkαIYRDLKPENLL (SEQ ID NO: 14)
Further, while there is conservation of sub-domain VIIa with respect to the equivalent motif in the other kinase families, the normally invariant D-F-G sequence of the PTK and serine/threonine families (motif VII) is replaced by the sequence D-P-G in motif VIIa of the JH2/KLD domain. The conservation of the precise sequence of sub-domain VI in the protein kinase sub-families appears to correlate with the substrate specificity of the kinase, and thus it is possible that this domain within the members of the JAK family of PTKs, may exhibit a substrate specificity other than that previously observed for other protein kinases.
A further sequence anomaly that may suggest a substrate variation lies within the putative ATP-binding site in the kinase-related domain (sub-domain Ia). This domain consists of the absolutely conserved -GXGXXG- in all PTKs described to date. However, in all known JAK family members, sub-domain Ia is replaced with -GXGXXT-. This glycine motif has now been defined as the ATP-binding site, with the first two glycine residues thought to bend around the nucleotide with the third glycine residue forming part of this loop. Substitution of the small side chain of glycine with the slightly larger threonine residue may disrupt the ATP-specific recognition, and confer some other substrate recognition. A viral mutation of the third glycine residue to a lysine in ν-SRC abolishes the transformation and catalytic activity of this oncogene (Verdaane and Varmus 1994). It is also noteworthy, that this glycine interacts sterically with the conserved phenylalanine and glycine of the sub-domain VII motif Asp-Phe-Gly in the catalytic domain of the Insulin Receptor (Hubbard et al., 1994). This conformation is involved in maintaining an open structure between the two lobes of the catalytic domain, and perhaps the altered glycine to threonine and phenylalanine to proline in KLD suggests an alternate structural requirement.
Certain other subtle differences exist in the normally consistent spacing between key motifs in KLD as compared with a PTK domain. For example, the spacing between both components of the ATP-binding site (Ia and IIa) is different for JAK1, JAK2, JAK3 and Tyk2 when compared with the broader protein kinase family. In JAK1 this spacing contains an extra 7 amino acids, JAK2 and JAK3 an extra 3 amino acids, and Tyk2, an extra 21 amino acids. Moreover, for JAK1, JAK2, Tyk2 and JAK3, the spacing between sub-domains VIa and VIIa in this region is also longer. Conversely, the distance between sub-domains VIIa and IXa in JAK1, Tyk2 and JAK3 is seven amino acids shorter that the corresponding region in the JH1 domain. It is worth noting that this sub-domain in the PTK domain contains the putative autophosphorylation tyrosine residue, while in each JAK family member, this tyrosine is not present in the KLD domain. The overall structure of this domain may be expected to be somewhat different from the catalytic domains of other members of the PTK and threonine/serine kinase families.
Regulation of the PTK Activity of JAK Kinases
Role of the KLD Domain
The tandem array of kinase domain and kinase-like domain is a defining feature of all members of the JAK family of PTKs. This fact, coupled with the high degree of conservation of the primary amino acid sequence of all members of this family, suggests that the role played by the KLD in the function of the JAK family of kinases is an important and evolutionarily conserved one. The presence of amino acid substitutions in key motifs within the KLD suggest that it is unlikely that this domain is a functional protein kinase. Indeed, attempts to demonstrate kinase activity form isolated purified KLD have so far proved to be impossible (Wilks et al., 1991) and it is often alternatively referred to as a pseudokinase domain.